Legionella pneumophila is a gram-negative bacterial species that infects a wide range of phagocytic host cells, including human macrophages and unicellular protozoa. Infection of alveolar macrophages results in the acute pneumonia known as legionnaires' disease. Infection of protozoa in natural or man-made environments is thought to provide a reservoir of the organism during outbreaks of legionnaires' disease. L. pneumophila inhibits phagosome fusion with lysosomes to survive and modifies the phagosome so that it becomes a favorable site for replication. A Type IV secretion system called the Icm/Dot system is absolutely required for intracellular survival and replication in mammalian and protozoan hosts but is dispensable for growth on bacteriologic media. A generally accepted hypothesis is that the Icm/Dot system translocates several effector molecules to host cells and that these effectors are directly responsible for interacting with host cell components and modifying organelle trafficking. During this funding period we: (i) identified three Legionella effector proteins (LepA, B, and C); (ii) showed that LepA and LepB are required for release of Legionella from protozoan host cells; and (iii) demonstrated translocation and cell contact dependent secretion of the Leps via the Icm/Dot system. In order to understand the molecular basis of L. pneumophila intracellular multiplication, we will: identify additional effectors, characterize the properties of the Lep proteins, and identify the host proteins that interact with them. To accomplish these goals we propose to: (i) Identify Legionella effectors based on their ability to disrupt endosomal trafficking in yeast. (ii) Identify Legionella proteins that are translocated to eukaryotic cells via the Icm/Dot system. (iii) Determine which host cell proteins interact with Legionella effector proteins. (iv) Test the hypothesis that the IcmO protein is the coupling factor of the Icm/Dot System. (v) Determine if the coiled:coil regions of the Lep proteins are required for translocation by the Icm/Dot system and/or for their function in host cells. [unreadable] [unreadable]